A racemate prodrug of leteprinim and methods of making and using the same

Abstract

The application provides a resiquimod prodrug and a preparation method and application thereof. The resiquimot prodrug comprises a carrier and a riboflavin derivative loaded on the carrier. The carrier is obtained by bonding R848-TK and mPEG-PHEA. b The application uses R848 NPs as a carrier to physically load a liposoluble riboflavin derivative, and is used for constructing an ultrasound-activated resiquimot prodrug. The riboflavin derivative is a liposoluble substance, can be activated under ultrasound conditions, and then generates 1 O2, is used for cutting the TK bond, thereby releasing the active drug R848. The application can effectively enhance the tumor selectivity of the drug and realize a time-space controllable precise treatment by preparing a more specific ultrasound-activated resiquimot prodrug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a rascimod prodrug, its preparation method, and its application. Background Technology

[0002] A prodrug is a biologically reversible derivative that is inactive in vivo but can effectively release an active drug after biotransformation. The term "prodrug" was first coined by Adrian Albert in 1958 to describe a compound that, after biotransformation, produces a pharmacologically active form. Rautio et al. further refined the definition of prodrugs. In recent years, prodrugs have been widely used due to their superior solubility, chemical stability, slow metabolism, and lower toxicity compared to the parent drug, becoming an important strategy for improving the pharmacokinetics and pharmacodynamics of active drugs and reducing their toxicity.

[0003] Prodrugs can be categorized into endogenous stimulus-responsive prodrugs and exogenous stimulus-responsive prodrugs based on their driving strategies. Endogenous stimulus-responsive prodrug activation is currently the most commonly approved strategy, typically utilizing enzymes or microenvironmental differences between diseased and normal tissues to activate the prodrug. For example, the thioclase (TK) bond is a commonly used ROS-responsive bond that can be cleaved by ROS and has been widely used in the construction of endogenous ROS-responsive prodrugs. However, most studies have shown that the limited differences between diseased and normal tissues often lead to insufficient drug specificity and selectivity, resulting in poor therapeutic effects and severe side effects. Therefore, designing a prodrug based on the TK bond with higher sensitivity, stronger selectivity, and the ability to release active drugs spatiotemporally and controllably on demand remains a major challenge.

[0004] Exogenous stimulus-responsive prodrugs offer a more promising prodrug strategy. Ultrasound, as a non-invasive physical radiation source, has been used in recent years as an exogenous stimulus to activate ultrasound-responsive prodrugs. Compared to endogenous stimulation, ultrasound activation has many advantages. First, ultrasound has extremely high tissue penetration, typically >10 cm. More importantly, ultrasound can achieve spatiotemporally controlled, site-specific activation with high specificity and is unaffected by individual differences. Over the past decade, ultrasound-mediated bond cleavage has been successfully applied in prodrug development, providing a more promising strategy for preparing highly tumor-selective, on-demand release drugs for spatiotemporally controlled precision treatment.

[0005] Resimod (R848), a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, has demonstrated good antitumor activity in multiple clinical trials, proving highly effective in stimulating immune responses and inhibiting tumor growth. However, due to the lack of tumor selectivity, R848 causes severe systemic toxicity after administration, hindering its further clinical application. Therefore, there is an urgent need to develop a prodrug with high specificity and selectivity that releases active R848 on demand at the lesion site, thereby reducing drug toxicity while achieving selective activation of the drug at the tumor site. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a rasimodil prodrug, its preparation method, and its application. The rasimodil prodrug reduces drug toxicity while enabling selective activation of the drug at the tumor site.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] This invention provides a rethimod prodrug comprising a carrier and a riboflavin derivative loaded on the carrier;

[0009] The carrier was obtained by bonding R848-TK with mPEG-b-PHEA.

[0010] Preferably, the riboflavin derivative is selected from any one or more of riboflavin tetrabutyrate, riboflavin tetraacetate, riboflavin tetrapropionate, riboflavin 2',3',4',5'-tetra(decanoate), riboflavin 5'-butyrate, riboflavin 5'-acetate, riboflavin adamantane, riboflavin 5'-(malonate), or riboflavin butyrate.

[0011] Preferably, the mass ratio of R848 to riboflavin derivative in the carrier is 1:(1~10).

[0012] Preferably, the rethimod prodrug has ultrasonic activation properties.

[0013] Secondly, the present invention provides a method for preparing the above-mentioned rascimodil prodrug, comprising the following steps:

[0014] After mixing the carrier and riboflavin derivative in the presence of an organic solvent, the organic solvent was removed, and the resulting product was self-assembled into micelles in water to obtain the retimote prodrug.

[0015] The carrier was obtained by bonding R848-TK with mPEG-b-PHEA.

[0016] Preferably, the organic solvent is selected from any one or more of chloroform, dichloromethane, or methanol.

[0017] Thirdly, the present invention also provides the application of rasimod prodrug in the preparation of antitumor drugs.

[0018] Fourthly, the present invention also provides an antitumor drug comprising the above-mentioned rasimod prodrug and a pharmaceutically acceptable excipient.

[0019] Preferably, the antitumor drug releases R848 from the rethimod prodrug under ultrasound conditions.

[0020] Preferably, the frequency of the ultrasound is 0.5~3 MHz; the power is 0~3 W / cm². 2 .

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention provides a rasimodil prodrug comprising a carrier and a riboflavin derivative loaded on the carrier. The carrier is obtained by bonding R848-TK with mPEG-b-PHEA. This invention uses R848 NPs as a carrier to physically support a lipid-soluble riboflavin derivative for constructing an ultrasound-activated rasimodil prodrug. The riboflavin derivative, being a lipid-soluble substance, can be activated under ultrasound conditions, thereby producing… 1 O2 is used to cleave the TK bond, thereby releasing the active drug R848. This invention, by preparing a more specific ultrasound-activated rasimod prodrug, can effectively enhance the tumor selectivity of the drug, achieving spatiotemporally controllable precision treatment. Attached Figure Description

[0023] Figure 1 A comparison of the fracture rates of TK-PrOH under different sound-sensing agents;

[0024] Figure 2 This is a schematic diagram of the synthesis process of R848 / TBR NPs in Example 3;

[0025] Figure 3 A color comparison chart of R848 NPs aqueous solution and R848 / TBR NPs solution;

[0026] Figure 4 The UV-Vis absorption spectrum of R848 / TBR NPs in Example 3;

[0027] Figure 5 The hydrated particle size distribution of R848 / TBR NPs in Example 3 is shown.

[0028] Figure 6 The UV-Vis absorption spectra of ICG blended with different concentrations of R848 / TBR NPs after ultrasonication are shown.

[0029] Figure 7 This is a schematic diagram of drug administration to mice in Application Example 1;

[0030] Figure 8 Tumor inhibition curves for different groups;

[0031] Figure 9 Tumor weight maps for different groups;

[0032] Figure 10 Figures showing the body weights of mice in different groups. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Because R848 alone lacks tumor selectivity and causes severe systemic toxicity after administration, it has failed to achieve further clinical application. Therefore, this application aims to prepare a prodrug that releases active R848 on demand at the lesion site, thereby reducing drug toxicity while achieving selective activation of the drug at the tumor site.

[0035] Based on this, the present invention provides a rethimod prodrug comprising a carrier and a sonicating agent loaded on the carrier.

[0036] In this invention, the carrier is R848 NPs, which are obtained by bonding R848 and mPEG-b-PHEA to a linker containing a ROS-responsive thioclase (TK) bond via amide condensation and esterification reactions, respectively. Specific preparation methods can be found in patent 202311219082.4. To avoid complexity, further details are omitted here.

[0037] In preliminary experiments, this invention investigated the sonication of several types of sonic saturators (including hematoporphyrin methyl ether (HMME), Bengal rose red (RB), doxorubicin (DOX), IR780, riboflavin (Rf), and indocyanine green (ICG)) with the substrate TK-PrOH, and analyzed the substrate breakage rate. The results showed that the substrate exhibited different breakage rates after sonication with different types of sonic saturators, indicating that different types of sonic saturators do indeed affect the TK bond breakage efficiency. Among the six sonic saturators, Rf showed the highest substrate breakage rate after sonication, therefore, this invention selected Rf for subsequent experiments. However, Rf itself is a water-soluble substance, making it difficult to achieve high loading efficiency. Therefore, to achieve higher loading efficiency, this invention preferably uses a riboflavin derivative, namely riboflavin ester, to construct a sonication-activated rasimod prodrug, utilizing its lipid solubility, to precisely release the active drug R848 while achieving a high loading rate.

[0038] In some embodiments of the present invention, the riboflavin derivative is selected from any one or more of riboflavin tetrabutyrate, riboflavin tetraacetate, riboflavin tetrapropionate, riboflavin 2',3',4',5'-tetra(decanoate), riboflavin 5'-butyrate, riboflavin 5'-acetate, riboflavin adamantane, riboflavin 5'-(malonate), or riboflavin butyrate, preferably riboflavin tetrabutyrate, also known as riboflavin tetrabutyrate, abbreviated as TBR.

[0039] It should be noted that too low a amount of riboflavin derivative may lead to insufficient TK bond cleavage, resulting in inadequate release of R848 and reduced drug efficacy. Conversely, too high a amount may require further optimization of the preparation method to ensure high loading efficiency. Therefore, the preferred mass ratio of R848 to riboflavin derivative in the carrier is 1:(1~10), more preferably 1:(3~5).

[0040] This invention uses R848 NPs as a carrier to physically support a lipid-soluble riboflavin derivative for the construction of an ultrasound-activated rasimod prodrug. The riboflavin derivative, being a lipid-soluble substance, can be activated under ultrasound conditions, thereby producing… 1 O2 is used to cleave the TK bond, thereby releasing the active drug R848. This invention, by preparing a more specific ultrasound-activated rasimod prodrug, can effectively enhance the tumor selectivity of the drug, achieving spatiotemporally controllable precision treatment.

[0041] The present invention also provides a method for preparing the above-mentioned rascimodil prodrug, which includes the following steps:

[0042] After mixing the carrier and riboflavin derivative in the presence of an organic solvent, the organic solvent was removed, and the resulting product was self-assembled into micelles in water to obtain the retimote prodrug.

[0043] The organic solvent mentioned above is selected from any one or more of chloroform, dichloromethane or methanol, preferably chloroform.

[0044] In some embodiments of the present invention, it is preferable to mix the carrier and the riboflavin derivative in the presence of an organic solvent, then remove the organic solvent under vacuum conditions. After solvent removal, a uniform thin film is formed on the wall of the container.

[0045] Then, according to the present invention, water is added to hydrate the film, and micelles are formed through self-assembly, i.e., retinoic acid prodrug. The water is preferably deionized water. The present invention does not have a particular limitation on the amount of water used; hydration of the film is sufficient.

[0046] The present invention also provides the application of the above-mentioned rasimod prodrug in the preparation of antitumor drugs.

[0047] The present invention also provides an antitumor drug comprising the above-mentioned rasimod prodrug and a pharmaceutically acceptable excipient. The excipient may be a substance well known to those skilled in the art.

[0048] The present invention also provides a method of using an antitumor drug, which includes the following steps:

[0049] After the anti-tumor drug is injected into the body, it is subjected to ultrasound treatment.

[0050] In some specific embodiments of the present invention, the tumor site is subjected to ultrasound treatment 6 hours after the above-mentioned drug is injected in vivo.

[0051] The frequency of the ultrasonic treatment is 0.5~3 MHz, preferably 1 MHz; the power is 0~3 W / cm². 2 The preferred value is 1~2W / cm 2 The duty cycle of the ultrasonic treatment is preferably 50%.

[0052] In practical application, this invention is preferably administered in two-day courses, for a total of three consecutive courses. Testing showed that the anti-tumor drug exhibited a significant tumor inhibition rate under ultrasound treatment, reaching 98.1%.

[0053] To further illustrate the present invention, the following embodiments provide a detailed description. The experimental materials used in the following embodiments of the present invention are all commercially available products.

[0054] Example 1

[0055] This embodiment investigated in vitro the cleavage efficiency of the substrate TK-PrOH containing TK bonds at different ultrasonic frequencies, ultrasonic powers, and ultrasonic times in the absence of a sound sensitizer. The specific method is as follows:

[0056] The substrate TK-PrOH was fully dissolved in a mixed solution of methanol and water, and then the solution was sonicated under different conditions, as shown in Table 1. Analysis... 1 The integral of the -CH3 peak at 1.48 ppm in the 1H NMR spectrum is used to determine the fragmentation rate of the substrate TK-PrOH.

[0057] The results are shown in Table 1 (wherein, the BLANK group represents the NMR integral data of TK-PrOH in aqueous solution; the H2O2 group and groups 1-12 all represent the NMR integral data in 100 μM hydrogen peroxide solution):

[0058] Table 1

[0059]

[0060] The results showed that, with fixed ultrasonic frequency and power, the degree of substrate breakage was positively correlated with ultrasonic time; with fixed ultrasonic frequency and time, the degree of substrate breakage was positively correlated with ultrasonic power; and with fixed ultrasonic power, the degree of breakage was negatively correlated with ultrasonic time. In short, long-duration, high-power low-frequency ultrasound is more conducive to the breakage of TK bonds (Table 1). However, in the absence of a sonicating agent, all substrates exhibited low breakage efficiency.

[0061] Therefore, the present invention further incorporates a sound-sensitive agent, as detailed in Example 2.

[0062] Example 2

[0063] This embodiment investigates the effects of different types of sonic sensitizers (including: hematoporphyrin methyl ether (HMME), Bengal rose red (RB), doxorubicin (DOX), IR780, riboflavin (Rf), and indocyanine green (ICG)) on the TK bond breaking efficiency. The specific methods are as follows:

[0064] The aforementioned types of acoustic sensitizers were co-blended with the substrate TK-PrOH and sonicated. The concentrations of both the acoustic sensitizers and the substrate TK-PrOH were 200 μM. The sonication conditions were 1 MHz and 2 W / cm². 2 The study was conducted at 10 min with a 50% duty cycle, and the fracture rate of the substrate was analyzed.

[0065] Test results are as follows Figure 1 As shown in Table 2:

[0066] Table 2

[0067]

[0068] As shown in Table 2, the substrates exhibited different fracture rates after being blended with different types of sonicators and sonicated. This indicates that different types of sonicators do indeed affect the fracture efficiency of TK bonds. Among the six sonicators mentioned above, Rf showed the highest substrate fracture rate after sonication. Figure 1 Therefore, this invention uses Rf for subsequent experiments.

[0069] Example 3

[0070] This embodiment provides a rasimod prodrug (abbreviated as: R848 / TBR NPs), the synthesis process of which is shown in the schematic diagram below. Figure 2 As shown, the specific method is as follows:

[0071] First, R848 nanoparticles, abbreviated as R848 NPs, were prepared according to patent 202311219082.4. The specific structure of the R848 NPs is shown in Formula 2:

[0072]

[0073] Formula 2;

[0074] Then, R848 NPs (100 mg) and riboflavin tetrabutyrate ester (TBR, 30 mg) were fully dissolved in CHCl3 (10 mL), and the solvent was removed under vacuum. A uniform thin film formed on the surface of the resulting solid product. Deionized water was then added to hydrate the film, and micelles were formed through self-assembly. The micelles were filtered through a 0.22 μm aqueous filter membrane to remove insoluble matter, and then lyophilized for 48 h to obtain the sonicated rasimodil prodrug (R848 / TBR NPs).

[0075] In this invention, the aqueous solution of the R848 NPs is colorless and transparent. After the introduction of TBR, the resulting R848 / TBR NPs solution is pale yellow. Figure 3 ).

[0076] The successful synthesis of R848 / TBR NPs was confirmed by UV-Vis absorption spectroscopy. Characteristic peaks of R848 appeared at 313 nm and 327 nm, while characteristic peaks of TBR appeared at 375 nm and 444 nm. The calculated molar ratio of R848 to TBR was 1:1.5. Figure 4 ).

[0077] Furthermore, the hydrated particle size of R848 NPs was measured to be approximately 170 nm by DLS. Figure 5 ).

[0078] This invention also uses ICG as 1 O2 probes were used to detect R848 / TBR NPs in vitro. 1 O2 generation capacity: UV results showed that after blending with different concentrations of R848 / TBR NPs and sonicating, the UV absorbance of ICG decreased significantly, proving that R848 / TBR NPs can effectively generate O2. 1 O2 ( Figure 6 ).

[0079] Application Example 1

[0080] In this application example, the prepared ultrasound-activated rasimod prodrug (R848 / TBR NPs) was used in combination with ultrasound to study its antitumor effect in a CT26 tumor model.

[0081] To construct a CT26 subcutaneous tumor model, CT26 tumor cells were inoculated into the abdomen of BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). When the tumor volume reached approximately 150 mm², CT26 tumor cells were introduced. 3 Mice were randomly divided into 9 groups: PBS, US, TBR, TBR+US, R848 NPs, R848 / TBR NPs, R848 NPs+US, R848 NPs+TBR+US, and R848 / TBR NPs+US, with 5 mice in each group. Each mouse in each group was injected via tail vein every other day (0, 2, 4 days) with either R848 NPs (eq.to R848 1.5 mg / kg), R848 / TBR NPs (eq.to R848 1.5 mg / kg), or TBR (4.8 mg / kg). Specifically: mice in the PBS group were injected with PBS (200 μL per mouse); mice in the TBR and TBR+US groups were injected with TBR; mice in the R848 NPs and R848 NPs+US groups were injected with R848 NPs; mice in the R848 NPs+TBR+US group were injected with a mixture of R848 NPs and TBR; and mice in the R848 / TBR NPs and R848 / TBR NPs+US groups were injected with R848 / TBR NPs. Six hours after the tail vein injection of the above drugs, the tumor sites in the groups containing US were subjected to ultrasound therapy (1... MHz, 2 W / cm 2 50% duty cycle, 2 min), one course of treatment every two days, for a total of 3 consecutive courses ( Figure 7 ).

[0082] Tumor volume was recorded every two days. The results showed that the PBS group exhibited the fastest tumor growth rate, reaching approximately 1500 mm² on day 12.3 R848 / TBR NPs+US showed a significant tumor inhibition rate, with a tumor inhibition rate of up to 98.1%. Figure 8 Mice were euthanized on day 12, and tumors were collected and weighed. A significant reduction in tumor weight was observed in mice treated with R848 / TBRNPs+US. Figure 9 ), and the mice did not experience significant weight loss during the treatment process. Figure 10 ).

[0083] The above results indicate that the R848 / TBR NPs+US group can significantly inhibit CT26 tumor growth, mainly because TBR can be activated under ultrasound conditions, producing a large number of [tumors / materials]. 1 O2 induces tumor cell apoptosis and simultaneously cleaves TK bonds to release R848, effectively inhibiting tumor growth.

[0084] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A resimod prodrug, characterized in that, Includes a carrier and a riboflavin derivative loaded on the carrier; the riboflavin derivative is selected from riboflavin tetrabutyrate; The carrier was obtained by bonding R848-TK with mPEG-b-PHEA; The mass ratio of R848 to riboflavin derivative in the carrier is 1:(1~10).

2. The resimilar prodrug according to claim 1, characterized in that, The retimote prodrug has ultrasound-activated properties.

3. A method for preparing a rethimod prodrug as described in claim 1 or 2, characterized in that, Includes the following steps: The carrier and riboflavin derivative were mixed in the presence of an organic solvent, the organic solvent was removed, and the resulting product was self-assembled into micelles in water to obtain retimote prodrug; the riboflavin derivative was selected from riboflavin tetrabutyrate. The carrier was obtained by bonding R848-TK with mPEG-b-PHEA; The mass ratio of R848 to riboflavin derivative in the carrier is 1:(1~10).

4. The preparation method according to claim 3, characterized in that, The organic solvent is selected from any one or more of chloroform, dichloromethane, or methanol.

5. The use of the rasimod prodrug according to claim 1 or 2, or the rasimod prodrug prepared by the preparation method according to claim 3 or 4, in the preparation of antitumor drugs, characterized in that, The tumor is colon cancer.

6. An antitumor drug, characterized in that, Includes the retimote prodrug as described in claim 1 or 2, or the retimote prodrug prepared by the preparation method according to claim 3 or 4, and pharmaceutically acceptable excipients.

7. The antitumor drug according to claim 6, characterized in that, The antitumor drug releases R848 from the rethimod prodrug under ultrasound conditions.

8. The antitumor drug according to claim 7, characterized in that, The frequency of the ultrasound is 0.5~3 MHz; the power is 0~3 W / cm². 2 .

Images